. G proteins are vital to many physiological processes such as vision, hormone response, olfaction, immune responses, and development. The G protein, Galpha2, has been shown to regulate cAMP signaling, chemotaxis, and differentiation in developing Dictyostelium discoideum. The chemotaxis pathway has been linked to the activation of guanylyl cyclase. In vivo, extracellular cAMP binds to a cell surface, G protein-coupled receptor, which leads to a rapid increase in cGMP in the cell. Cyclic-AMP-mediated activation of guanylyl cyclase and chemotaxis are dependent on Galpha2. In vitro, guanylyl cyclase is activated by GTPgammaS. This compound can also activate guanylyl cyclase in Galpha2-minus cells, suggesting that Galpha2 is indirectly required for the stimulation of guanylyl cyclase. Two approaches will be employed to identify the pathway intermediates required for the activation of guanylyl cyclase via Galpha2 in Dictyostelium. In the first, restriction enzyme-mediated integration (REMI) mutagenesis will be performed to identify genes required for Galpha2-dependent activation of guanylyl cyclase. Aggregation defective mutants will be selected and screened to identify those defective in chemotaxis. Additional screening will be used to specifically select mutants that are defective between cAMP receptor-Galpha2 interaction and guanylyl cyclase. The disrupted gene will be isolated from the selected mutants and identified by DNA sequencing. In the second approach, the two-hybrid system will be used to identify proteins that directly interact with Galpha2. This approach will identify the downstream effectors of Galpha2.